Reagents and methods for replication, transcription, and translation in semi-synthetic organisms

Abstract

Disclosed herein are compositions, methods, cells, engineered microorganisms, and kits for increasing the production of proteins or polypeptides comprising one or more unnatural amino acids. Further provided are compositions, cells, engineered microorganisms, and kits for increasing the retention of unnatural nucleic acids encoding the unnatural amino acids in an engineered cell, or semi-synthetic organism.

What is claimed is: 
     
       1. A nucleobase of the structure: 
       
         
           
           
               
               
           
         
         wherein: 
         each X is independently carbon or nitrogen; 
         R 2  is present when X is carbon and is independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, or azide group; 
         Y is sulfur, oxygen, or selenium; and 
         E is sulfur; 
         wherein the wavy line indicates a point of bonding to a ribosyl, deoxyribosyl, or dideoxyribosyl moiety, wherein the ribosyl, deoxyribosyl, or dideoxyribosyl moiety is optionally modified at the 2′ position, at the 3′ position, or at the 5′ position, wherein the ribosyl, deoxyribosyl, or dideoxyribosyl moiety is in free form, is connected to a mono-phosphate, diphosphate, triphosphate, α-thiotriphosphate, β-thiotriphosphate, or γ-thiotriphosphate group, or is included in a polynucleotide, optionally wherein the polynucleotide is an RNA, DNA, bicyclic nucleic acid, linked nucleic acid, peptide nucleic acid (PNA), locked nucleic acid (LNA), or a phosphorothioate-containing nucleic acid. 
       
     
     
       2. The nucleobase of  claim 1 , wherein X is carbon and/or Y is sulfur. 
     
     
       3. The nucleobase of  claim 1 , which has the structure 
       
         
           
           
               
               
           
         
       
     
     
       4. The nucleobase of  claim 1 , which is bound to a complementary base-pairing nucleobase to form an unnatural base pair (UBP), wherein the complementary base-pairing nucleobase is selected from: 
       
         
           
           
               
               
           
         
       
     
     
       5. A double stranded oligonucleotide duplex wherein a first oligonucleotide strand comprises the nucleobase of  claim 1 , and a second complementary oligonucleotide strand comprises a complementary base-pairing nucleobase in a complementary base-pairing site thereof. 
     
     
       6. The double stranded oligonucleotide duplex of  claim 5 , wherein the first oligonucleotide strand comprises 
       
         
           
           
               
               
           
         
       
       and the second strand comprises a complementary base pairing nucleobase selected from: 
       
         
           
           
               
               
           
         
       
       in a complementary base-pairing site thereof. 
     
     
       7. A DNA comprising a nucleobase having the structure 
       
         
           
           
               
               
           
         
       
       and a complementary base-pairing nucleobase having the structure 
       
         
           
           
               
               
           
         
       
       or a nucleobase having the structure 
       
         
           
           
               
               
           
         
       
       and a complementary base-pairing nucleobase having the structure